PROJECT SUMMARY Iron plays a central role in cell proliferation and has been implicated in several aspects of cancer biology. Malignant cells harbor an altered iron metabolism aimed at increasing iron acquisition and retention, thereby supporting rapid proliferation rates. Iron scavengers, including several clinical chelators for the treatment of iron overload, exhibit antiproliferative properties, albeit in the presence of narrow therapeutic windows and systemic toxicity in several cases. The higher demand for iron of cancer cells is now recognized as an important area of investigation and a therapeutic opportunity; however, currently available chelator systems are not designed specifically to target intracellular iron in malignant cells. This research program seeks to engineer contemporary approaches to iron chelation, particularly to improve our control of intracellular delivery and tumor selectivity as well as our understanding of the parameters correlated to iron deprivation in cell proliferation and malignancy. Under the first specific aim of the project, we build on our work on prochelator systems that are activated for iron coordination upon cell entry. Parameters affecting intracellular oxidative reactivity and toxicity will be assessed and tuned in several tridentate scaffolds. Within the second aim, tumor- targeting units are connected to prochelators designed to increase cancer selectivity. The study of a new class of glycoconjugate constructs takes advantage of the glucose avidity of malignant cells. In addition, the reactivity of acquired cysteine residues in oncogenic mutant proteins will be employed to activate prochelators in cancer cells carrying a specific mutation. Under the third aim of the project, the effects of the chelator systems on the cytosolic labile iron pool will be assessed through several spectroscopic methods. Their impact on iron homeostasis will be examined through the post-transcriptional regulation and expression of proteins involved in iron uptake, transport, and storage. Finally, our analyses of cell cycle, death and metabolic parameters will contribute to delineate the effective cellular susceptibility to our chelator systems. Breast, colon, and pancreatic cancer cell lines were selected for this study because the implication of iron in their progression and aggressive phenotypes is documented in cell studies, animal models, and clinical data. This study is innovative in its combination of principles of iron coordination chemistry with pro-drug and tumor- targeting approaches for intracellular iron binding. Motivated by the increased iron needs of all cancer phenotypes, this research program is poised to produce broadly applicable iron-binding strategies and fundamental information on their impact in cell cycle progression with the long-term goal of identifying new potential avenues for cancer treatment.